Brazzein encompasses a group of related sweet proteins extracted and purified from the fruit of the African plant, Pentadiplandra brazzeana. (Ming and Hellekant, FEBS Lett. 355: 106-108, 1994). Brazzein occurs in three naturally occurring forms, the amino acid sequences of which are known: Type I is 54 amino acids in length and has an N-terminal pyrrolidone carboxylic acid; Type II is 54 amino acids in length and has an N-terminal glutamine, and Type III is 53 amino acids in length and has an N-terminal aspartic acid (Assadi-Porter, et al., Arch. Biochem. Biophys. 376: 252-258, 2000). In addition to the natural forms of brazzein, variants of brazzein, i.e., mutated and recombinant proteins with varied sequences and properties, have been created by genetic engineering, such as those described in U.S. Pat. Nos. 5,326,580; 5,346,998; 5,527,555; 5,741,537; 7,153,535; 6,274,707 and in Jin, et al., Chemical Senses 28: 491-498; Assadi-Porter, et al., Arch. Biochem. Biophys. 376: 259-265, 2000; Assadi-Porter, et al., Chem. Senses 30 (Suppl. 1): i90-i91, 2005. Brazzein is a high-intensity natural sweetener, having an excellent taste profile, 400-500 times the sweetness of sucrose, and excellent stability at low pH and high temperatures.
Described below are methods, using food-grade methodology and yeast fermentation, for enhanced production and improved purification of a natural, high-quality brazzein protein that enable the commercial production of brazzein. This unique protein has applications as a sweetener in the baking, beverage, and table-top product industries.